Conjunctivitis and blepharitis are the most common ocular infective pathologies. Even if the infection has a self-limiting clinical course, a pharmacological treatment must be used to limit the diffusion of the infective event. The efficacy of an antibiotic therapy in the treatment of ocular infections is related to several factors, mainly the pharmaceutical form used and the bacterial susceptibility to the drug. It is sufficient to use wide-spectrum antibiotics that are effective on the pathogens that are commonly involved in ocular infections. As far as the pharmaceutical form is concerned, the antibiotic must guarantee a release of optimal pharmacological concentrations into the site of infection. In this context, the pharmacokinetic properties of a chosen drug play a key role. The concentration of the drug at the site of infection must be higher than the minimal inhibitory concentrations (MIC) of most common pathogens.
Among the ophthalmic antibiotics available, aminoglycosides are the drugs of choice in the treatment of ocular infections. This is because they have a wide antimicrobial spectrum, a rapid concentration-dependent bactericidal effect, a good clinical efficacy, and a low incidence of bacterial resistance. 1–4 Since its recent introduction into the European ophthalmic market, netilmicin has been used for the treatment of external ocular infections. Netilmicin is a semisynthetic derivative of sisomicin obtained by N-ethyl substitution into the first position of the 2-deoxystreptamine ring. This substitution provides additional protection against bacterial enzymes that deactivate tobramycin and gentamicin, which bear an amino group in that position.
According to clinical trial results, this new aminoglycoside is effective because of its activity on strains resistant to gentamicin-and tobramycin-, and on Staphylococcus aureus (MRSA) methicillin-resistant strains. 5,6 Additionally, clinical trials show that the side effects and contraindications for netilmicin local tolerance are similar to those for tobramycin and gentamicin.
A previous study on the kinetic of aminoglycosides on the ocular surface reported that tear film concentration of gentamicin sharply decreases below 0.1 mg/mL a few minutes after the topical application at a concentration of 3 mg/mL. 7
Because of the lack of data in the literature, the purpose of this study is to evaluate the residence time and the biologic half-life of netilmicin eye drops on the ocular surface, by determining its concentration in the tears of healthy volunteers. The investigation followed the criteria of previous studies regarding other classes of antibiotics. 8,9
MATERIALS AND METHODS
This study was carried out by a qualified doctor, P. Zola, in a private ambulatory, in accordance with the tenets of the Declaration of Helsinki. The investigation was conducted with 32 healthy volunteers; mean age of 35 years (range 12–68). Volunteers were enrolled after releasing informed consent. After enrollment, inclusion and the exclusion criteria were verified. Exclusion criteria were considered: ocular concomitant disorders (i.e. dry-eye or lachrymal film alterations); drug hypersensivity; concomitant topical antibiotic therapy; contact lenses wearers; pregnancy and/or nursing; noncooperation. Subjects were administered one drop of netilmicin 3 mg/mL in both eyes. The eye drops were placed in the inferior cul-de-sac. They were interviewed about tolerability and any adverse ocular reaction related to the drug administration was registered. The subjects underwent biomicroscopy and ophthalmoscopy examinations to verify the presence of objective side effects. After the examinations, each subject was assigned to one of four study groups. Tears were collected in each group at 5, 10, 20, and 60 minutes after the drug instillation, respectively.
The lachrymal collection was performed without topical anesthesia by using a 5-μL glass-capillary micropipette (Baxter, Pisa, Italy). The capillary was applied in the inferior lateral “cul-de-sac” until 0.5 μL was obtained. The lachrymal amount was placed in the analytic vials (Agilent, Milan, Italy) and kept refrigerated at 4°C until analyzed.
High-pressure liquid chromatography (HPLC) analysis was conducted on a Hewlett Packard HPLC (Hewlett Packard, Wilmington, DE, U.S.A.) system consisting of a 1090 pump equipped with UV/DAD detector and 1100 autosampler and chemstation. Data were collected and processed by HP Chemstation. A hypersil ODS column (5 μm, 100 × 2.1 mm I.D.) protected by a precolumn was used to separate netilmicin after derivatization with 1,2-phtalic dicarboxaaldehyde. The mobile phase was 150/800/50 V/V water, methanol, and aqueous solution obtained by dissolving 5 g heptane-sulfonic acid sodium salt in 50 mL glacial acetic acid; the flow was 0.5 mL/min. Netilmicin was detected at 330 nm with retention time of ca. 4 minutes.
Samples were prepared by adding 10 μl of o-phtalaldeide solution (10 mg were dissolved in 5 mL methanol, then 90 mL borate buffer [pH 10.4] and 0.2 mL thioglycolic acid were added) and 10 μL of methanol to the vial containing the sample. The mixture was heated at 60°C for 15 minutes, cooled to 4°C and injected in HPLC (7 μL). Reference standards were prepared using 5 μL of standard solution (240–6 ppm), instead of the tear.
A standard calibration curve was plotted with netilmicin sulphate reference standard, and netilmicin concentration was determined by substituting the sample peak area in the calibration curve equation. Sensitivity was 2 μg/mL. The mean and standard deviation were calculated at each collection for volunteers belonging to the same group.
The study was conducted with 32 volunteers who did not declare any adverse event or side effects. One volunteer was dismissed because of the difficulty to sample the tear. Of the remaining 31 volunteers, 15 were male and 17 were female. The sample's mean age was 37 years with a range from 31 to 68. Tears were sampled 5 minutes after administration of eye drops on 11 subjects, 10 minutes after administration on 10 subjects, 20 minutes after administration on 5 subjects and 60 minutes after administration on 6 subjects.
Determination of netilmicin concentration
The data collected in the experiment are shown in Figure 1. Mean values of netilmicin concentration are 256 ± 167 μg/ml at 5 minutes, 182 ± 140 μg/ml at 10 minutes, 94.0 ± 94 μg/ml at 20 minutes and 27.5 ± 14 μg/ml at 60 minutes.
The figure shows that data variability is greater at 5 and 10 minutes because of individual reaction to the instillation of the eye drop. Although these points are scattered, they are symmetrically distributed around the mean value. This enables us to calculate a best-fit curve through the mean values. The result is an exponential function as reported in equation : C = 263 × e −0.039t .
The first order kinetic of elimination with a correlation coefficient of 0.97 can be calculated from the exponential best-fit line. It can be extrapolated from equation  that the average netilmicin concentration immediately after instillation is 263 μg/mL corresponding to 8.9% of the eye drop concentration. The half-life of the drug in tears is 18 minutes.
Netilmicin good tolerability profile agrees with data referring to clinical studies carried out on adults and pediatric patients. 10,11
The capillary tears suction from the lower conjunctival sac has been found to be advantageous if compared with Schirmer strips, especially because it allows direct transfer of the tears in the measurement device. 12 It has been observed that the tear sampling could only be carried out on a single eye with a randomized criterion. The application of a capillary on the inferior “cul-de-sac” causes reflex tearing in the fellow eye and therefore dilution of the substance.
Three principal processes determine the permanence in tears of a topically applied drug: the drainage, the tear turnover and the absorption onto biologic materials (membranes, mucins, etc.).
Drainage is the faster process occurring within the first 2 minutes after instillation. Since the tear volume is 10 μL or less, the maximum volume of fluid that can be contained in the “cul-de-sac” without overflow is 30 μL. The eye drop volume is approximately 40 μL therefore the excess fluid instilled is rapidly removed by spillage from the conjunctiva sac until the tear fluid returns to its original volume. 13
The reflex tearing after the instillation of an irritating drug may produce up to 400 μL excess tears. Thus, dilution of 5–50 fold usually occurs in the first 2 minutes, depending on the irritating power of the substance. The dilution factor found in our study (11 folds) confirms the good clinical tolerability of netilmicin administered as eye drops.
One of the successful factors in the treatment of external ocular infection is the presence of pharmacological doses able to inhibit the survival of the pathogen in the affected site. The extrapolation of netilmicin concentration in tears permits the evaluation of the minimum timeframe in which the antibiotic exceeds the MIC 90 . Table 1 reports the MIC 90 for the most common microorganisms and the calculated residence times when the netilmicin decay curve crosses over the MIC 90 .
This study shows that a single netilmicin ocular administration in healthy volunteers determines pharmacological concentrations of the drug better than the MIC 90 of most important pathogens after 5, 10, 20, and 60 minutes after topical instillation. Netilmicin remains on the ocular surface 100× better than the MIC 90 , of ocular strains such as Staphylococcus aureus. These data are more relevant if we consider that gentamicin concentrations exceed the MIC 90 of gram-positive strains for only 20 minutes after topical administration. 5 Additionally, considering all cases, netilmicin follows a path of concentration that enables us to assume that concentration above the MIC 90 are ensured even for the subjects who exhibited low netilmicin concentration at 5 and 10 minutes. Extrapolation of the mean values curve establishes that netilmicin residence time in tears is sufficiently prolonged to exert its antimicrobial activity for more than 2 hours.
Finally, it has to be considered that one of the most important pharmacodynamic properties of aminoglycosides is the post antibiotic effect (PAE), which consists of a continued suppression of bacterial growth after the antibiotic concentration drops lower than the MIC. It has been demonstrated that aminoglycosides have a PAE of 1–8 hours both in vitro and in vivo for various gram-negative bacilli after the exposure to drug concentrations equal to 2–10 times the MIC. 2 Additionally, it has been shown that higher doses of amynoglicosides determine a longer PAE. This observation has important consequences in clinical practice, as it allows for longer dose intervals than the one predicted from MIC values alone.
Taking into consideration our results and the pharmacodynamic features (i.e. PAE) of aminoglycosides, we can conclude that netilmicin provides adequate concentration for challenging infections occurring on the ocular surface. This result is consistent with the current posology of four administrations per day. Additionally, netilmicin showed high compatibility with the tear environment.
Further studies will ascertain if more sophisticated methods of drug delivery can increase the residence of netilmicin in tears and, in the long rate, reduce the posology.
The authors are grateful to Miss Anna Longo who carried out the HPLC work.
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